Methods for detecting mutations using primer extension for detecting disease

ABSTRACT

Methods of the invention comprise assays for markers indicative of cancer, precancer, and other diseases or disorders. Assays of the invention are preformed on heterogeneous samples obtained from patients by non-invasive or minimally-invasive methods. Such assays may be employed alone or in combination with other disease screening techniques.

RELATED APPLICATIONS

[0001] This application is a continuation in part of U.S. applicationSer. No. 09/371,991 and U.S. application Ser. No. 09/468,670, whichclaims the benefit of 60/134,711, the disclosures of each of which isincorporated by reference herein.

FIELD OF THE INVENTION

[0002] The invention relates generally to methods of detecting cancer,precancer, or other diseases or disorders using nucleic acid markers.

BACKGROUND OF THE INVENTION

[0003] Numerous diseases are associated with disruptions in genomicstability. For example, sickle cell anemia, phenylketonuria, hemophilia,cystic fibrosis, and various cancers have been associated with one ormore genetic mutation(s). Cancer is thought to arise from a multi-stepprocess that typically involves multiple genetic mutations leading touncontrolled cell growth. Many cancers are curable if detected early intheir development. For example, colorectal cancers typically originatein the colonic epithelium, and are not extensively vascularized (andtherefore not invasive) during early stages of development. Thetransition to a highly-vascularized, invasive and ultimately metastaticcancer commonly takes ten years or longer. If the presence of cancer isdetected prior to extensive vascularization, surgical removal typicallyis an effective cure. However, colorectal cancer is often detected onlyupon manifestation of clinical symptoms, such as pain and bloody stool.Generally, such symptoms are present only when the disease is wellestablished, and often after metastasis has occurred. Similarly, withthe exception of the Pap smear for detection of pre-malignant cervicallesions, diagnostic screening methods for other types of cancer are bestat detecting established disease. Increased knowledge of the molecularbasis for disease has lead to a proliferation of screening assayscapable of detecting disease-associated nucleic acid mutations.

[0004] A variety of detection methods have been developed which exploitsequence variations in DNA using enzymatic and chemical cleavagetechniques. A commonly-used screen for DNA polymorphisms consists ofdigesting DNA with restriction endonucleases and analyzing the resultingfragments by means of Southern blots, as reported by Botstein et al.,Am. J. Hum. Genet., 32: 314-331 (1980) and White et al., Sci. Am., 258:40-48 (1988). Mutations that affect the recognition sequence of theendonuclease will preclude enzymatic cleavage at that site, therebyaltering the cleavage pattern of the DNA. Thus, a difference inrestriction fragment lengths is indicative of the presence of a mutationin the recognition sequence. A problem with this method (known asrestriction fragment length polymorphism mapping or RFLP mapping) is itsinability to detect a mutation outside of the recognition sequence andwhich, consequently, does not affect cleavage with a restrictionendonuclease. One study reported that only 0.7% of the mutationalvariants estimated to be present in a 40,000 base pair region of humanDNA were detected using RFLP mapping. Jeffreys, Cell, 18: 1-18 (1979).

[0005] Single-base mutations have been detected by differentialhybridization techniques using allele-specific oligonucleotide probes.Saiki et al., Proc. Natl. Acad. Sci., 86: 6230-6234 (1989). Mutationsare identified on the basis of the higher thermal stability of theperfectly-matched probes as compared to mismatched probes. Disadvantagesof this approach for mutation analysis include the requirement foroptimization of hybridization for each probe, and the limitationsimposed by the nature of the mismatch and the local sequence on thedegree of discrimination of the probes. In practice, tests based only onparameters of nucleic acid hybridization function poorly when thesequence complexity of the test sample is high (e.g., in a heterogeneousbiological sample). This is due partly to the small thermodynamicdifferences in hybrid stability generated by single nucleotide changes.Therefore, nucleic acid hybridization is generally combined with someother selection or enrichment procedure for analytical and diagnosticpurposes.

[0006] Recently, a number of genetic mutations, including alterations inthe BAT-26 segment of the MSH2 mismatch repair gene, the p53 gene, theKras oncogene, and the APC tumor suppressor gene have been associatedwith the multi-step pathway leading to cancer. The BAT-26 segmentcontains a long poly-A tract. In certain cancers, a characteristic 5base pair deletion occurs in the poly-A tract. Detection of thatdeletion may provide diagnostic information. For example, it has beensuggested that mutations in those genes might be a basis for molecularscreening assays for the early stages of certain types of cancer. Seee.g., Sidransky, et al., Science, 256: 102-105 (1992). Attempts havebeen made to identify and use nucleic acid markers that are indicativeof cancer. However, even when such markers are found, using them toscreen patient samples, especially heterogeneous samples, has provenunsuccessful either due to an inability to obtain sufficient samplematerial, or due to the low sensitivity that results from measuring onlya single marker. For example, simply obtaining adequate human DNA fromone type of heterogeneous sample (stool) has proven difficult. SeeVilla, et al., Gastroenterol., 110: 1346-1353 (1996) (reporting thatonly 44.7% of all stool specimens, and only 32.6% of stools from healthyindividuals produced sufficient DNA for mutation analysis). Otherreports in which adequate DNA has been obtained have reported lowsensitivity in identifying a patient's disease status based upon asingle cancer-associated mutation. See Eguchi, et al., Cancer, 77:1707-1710 (1996) (using ap53 mutation as a marker for cancer).

[0007] Therefore, there is a need in the art for high-sensitivity,high-specificity assays for the detection of molecular indicia ofcancer, pre-cancer, and other diseases or disorders, especially inheterogeneous samples. Accordingly, the invention provides methods fordetecting deletions in genomic regions, such as BAT-26 and others, whichmay be associated with disease.

SUMMARY OF THE INVENTION

[0008] Methods of the invention provide assays for identification of amutation in a genomic region suspected to be indicative of disease. Ingeneral, methods of the invention comprise annealing a primer upstreamof a region in which, for example, a deletion is suspected to occur,extending the primer through the region, terminating extension at aknown end-point, and comparing the length and/or weight of the extendedprimer with that of an extended primer from the corresponding wild-type(non-affected) region or a molecular weight standard (either known orrun in parallel). Also according to the invention, assays describedherein are combined with invasive detection methods to increasesensitivity of detection.

[0009] Methods of the invention further provide for the determination ofwhether a target point mutation is present at a genetic locus ofinterest. In one embodiment, the invention comprises contacting anucleic acid in a biological sample with a primer that is complementaryto a portion of a genetic locus, extending the primer in the presence ofa labeled nucleotide that is complementary to a target nucleotidesuspected to be present at the target position. The primer is furtherextended in the presence of a terminator nucleotide that iscomplementary to a nucleotide downstream from the target nucleotide, butis not complementary to the target nucleotide, thereby generating anextension product. The presence of a labeled nucleotide in the extensionproduct is indicative of the presence of the target point mutation atthe genetic locus.

[0010] In addition, methods of the invention provide for theidentification of a target single nucleotide polymorphic variant presentat a genetic locus of interest. In one embodiment, the method comprisescontacting a nucleic acid in a biological sample with a primer,extending the primer in the presence of at least a first and a seconddifferentially labeled nucleotide, the first labeled nucleotide beingcomplementary to a first nucleotide suspected to be present at saidtarget position, the second labeled nucleotide being complementary to asecond nucleotide alternatively suspected to be present at the targetposition. The primer is further extended in the presence of a terminatornucleotide that is complementary to a nucleotide downstream from thetarget position, wherein the terminator nucleotide is not complementaryto the first or second nucleotides, thereby generating an extensionproduct. The identity of the labeled nucleotide present in the extensionproduct is indicative of the identity of the target single nucleotidepolymorphic variant present at the genetic locus.

[0011] In yet another embodiment, the first labeled nucleotide comprisesa first acceptor molecule and the second labeled nucleotide comprises asecond acceptor molecule with the first acceptor molecule beingdifferent from the second acceptor molecule. Also, the primer comprisesa donor molecule being capable of activating the first and secondacceptor molecules so as to produce a first and a second detectablesignal.

[0012] Furthermore, the methods of the invention provide thedetermination of whether a target single nucleotide polymorphic variantis present at a genetic locus of interest. For example, the methodcomprises contacting a nucleic acid in a biological sample with aprimer, extending the primer in the presence of a labeled nucleotidethat is complementary to a nucleotide suspected to be present at thetarget position, further extending the primer in the presence of aterminator nucleotide that is complementary to a nucleotide downstreamfrom the target nucleotide, wherein the terminator nucleotide is notcomplementary to the target nucleotide, thereby to produce an extensionproduct; and determining whether the labeled nucleotide is present inthe extension product, thereby determining whether the target singlenucleotide polymorphic variant is present at the genetic locus.

[0013] Moreover, the methods of the invention provides thequantification of the number of a nucleic acid having a targetnucleotide present at a genetic locus of interest. In general, themethod comprises contacting a nucleic acid in a biological sample with aprimer, extending the primer in the presence of a labeled nucleotidethat is complementary to target nucleotide, further extending the primerin the presence of a terminator nucleotide that is complementary to anucleotide downstream from the target nucleotide, wherein the terminatornucleotide is not complementary to the target nucleotide, thereby toform an extension product, and enumerating the number of extensionproducts that comprise the labeled nucleotide, thereby determining thenumber of nucleic acids having the target nucleotide at the geneticlocus.

[0014] In preferred embodiments, an extended primer produced in methodsof the invention is labeled downstream of the region suspected tocontain a mutation. In a preferred embodiment, the comparative lengthand/or molecular weight of the extended primer is determined by gelelectrophoresis or mass spectroscopy. Also in a preferred embodiment,the region suspected to contain the mutation comprises a poly-nucleotidetract in which a deletion is suspected to occur, and the sequenceimmediately downstream of the region is known and does not repeat anucleotide species present in the polynucleotide tract. Preferably, thepolynucleotide tract comprise three, two, or preferably one, species ofnucleotide as explained in detail below. Methods of the invention retainthe specificity of primer extension assays while increasing theirsensitivity by reducing background due to premature termination of theextension reaction. Therefore, methods of the invention provide a highlysensitive and highly specific assay for detecting a small amount ofmutant nucleic acid in a heterogeneous sample of predominantly wild-typenucleic acid.

[0015] Methods of the invention provide screening assays for thedetection of a deletion in a region of the genome comprising at leastone, but no more than three, species of nucleotide, and that ischaracterized by having a sequence for primer hybridization immediatelyupstream, and a sequence immediately downstream that does not contain anucleotide present in the region suspected to be deleted. In a preferredembodiment, methods of the invention comprise selecting a nucleic acidhaving a known wild-type sequence and having a region (the deletion ofwhich is suspected in disease) comprising at most three different typesof nucleotides; hybridizing an oligonucleotide primer, or pair ofoligonucleotide primers, immediately upstream of the target region;extending the primer by using a polymerase in the presence of thenucleotide bases that are complementary to the nucleotide bases of thetarget region, thereby to form a primer extension product; furtherextending the primer extension product in the presence of a labelednucleotide that is complementary to a nucleotide base downstream fromthe target region, but not complementary to a nucleotide base within thetarget region; and determining the size of the extension productcompared to a standard (e.g., a wild-type product or a molecular weightstandard).

[0016] For purposes of the present invention a “mutation” includes adeletion, addition, substitution, transition, transversion,rearrangement, and translocation in a nucleic acid, as well as a loss ofheterozygosity. A loss of heterozygosity is a form of mutation in whichall or a portion of one allele is deleted. Also for purposes of thepresent invention, the terms “markers”, “targets”, and “mutations”include nucleic acid (especially DNA) mutations, as well as othernucleic acid indicia useful in methods of the invention, such asspecific alleles and single nucleotide polymorphism variants. Suchindicia also include the amount of amplifiable nucleic acid in a sample,the integrity and/or length of nucleic acids in a sample, the ratio ofhigh integrity nucleic acids (greater than about 200 base pairs) to lowintegrity nucleic acids (less than about 200 base pairs), and any othernucleic acid variations that differ between patients with cancer anddisease-free patients.

[0017] In a preferred embodiment, the target region in which a deletionis suspected to occur is greater than five nucleotides long, and/or thedeletion is greater than three nucleotides long. In a preferredembodiment, the primer extension reactions are cycled by varying thereaction temperature through successive annealing, extending anddenaturing temperatures. Preferably, the molecular weight standard isthe wild-type extension product, or one that corresponds to the expectedsize for the extension product from the wild-type nucleic acid template.The presence of an extension product smaller than the molecular weightstandard is indicative of the presence of a deletion in the targetregion of the nucleic acid template. In a preferred embodiment, theprimer extension product is terminated by incorporating a terminatornucleotide that is complementary to a nucleotide downstream from thetarget region in a wild type nucleic acid, but not complementary to anyof the nucleotides of the target region. In a more preferred embodiment,the labeled nucleotide and the terminator nucleotide are the same. In analternative embodiment, more than one labeled nucleotide base isincorporated into the extension product prior to incorporation of theterminator nucleotide. Preferably, the nucleotides incorporated duringextension through the region suspected of containing a deletion areunlabeled. However, if those nucleotides are labeled, they arepreferably distinguishable from the labeled nucleotide that isincorporated at the 3′ end of the extension product.

[0018] In a preferred embodiment, methods of the invention comprisedetecting a nucleic acid mutation in a biological sample, such as stool,urine, semen, blood, sputum, cerebrospinal fluid, pus, or aspirate, thatcontains a heterogeneous mixture of nucleic acid having a deletion inthe target region and wild type nucleic acid. Such a mutation in thetarget region may be present in only about 1-5% of the nucleic acidmolecules having the target region. To increase the sensitivity of theassay, the sample may comprise a polymerase chain reaction product.Method of the invention are particularly useful in analyzing a deletionin the target region that is indicative of the presence of cancerous orprecancerous tissue in such a biological sample, including colorectalcancer or precancer detection in stool.

[0019] In another embodiment, methods of the invention comprise furtherextending the primer extension product in the presence of labeled andunlabeled nucleotides, the nucleotides being of the same type (i.e., A,T, C, or G) and being complementary to one or more nucleotide downstreamfrom the target region but not complementary to a nucleotide within thetarget region. In one embodiment, the ratio of the labeled nucleotide tounlabeled nucleotide is 1:1. Methods of the invention may also includeincorporating more than one monomer of the labeled nucleotide orunlabeled nucleotide into the extension product.

[0020] In another embodiment, methods of the invention comprisedetecting a deletion in a sample by selecting a nucleic acid with aknown wild-type sequence and having a target region suspected ofcontaining a deletion, wherein the target region contains at most threedifferent types of nucleotide bases selected from the group consistingof dGTP, dATP, dTTP, and dCTP; hybridizing an oligonucleotide primer toa region upstream of said target region in a nucleic acid sample;contacting said hybridized oligonucleotide primer with an extensionreaction mixture comprising: i) nucleotides which are complementary tothe nucleotides in the target region, ii) a labeled nucleotide which iscomplementary to a nucleotide found downstream from the target region,but which is not complementary to any nucleotide base found within thetarget region, and iii) a terminator nucleotide which is complementaryto a nucleotide found downstream from the target region, but which isnot complementary to any nucleotide found in the target region;extending the hybridized oligonucleotide primer to generate a labeledextension product; and comparing the size of the labeled extensionproduct to a molecular weight standard, wherein a labeled extensionproduct smaller than the molecular weight standard is indicative of thepresence of a deletion in the target region.

[0021] In another embodiment, methods of the invention comprise singlebase extension assays that detect low-frequency molecular events in abiological sample. Methods for detecting low-frequency molecular eventsin a biological sample are provided in U.S. Pat. No. 4,683,202, thedisclosure of which is incorporated by reference herein. Specificnucleic acids may be detected in a biological sample with both highsensitivity and high specificity. In general, methods of the inventioncomprise performing a single-base extension reaction utilizing donor andacceptor molecules which interact to produce a detectable signal.

[0022] The nucleotides comprise an acceptor molecule which interactswith a donor molecule on the primer when in close proximity and thusfacilitates detection of the extended primers, or extended short firstprobes in an extension reaction. The donor and acceptor molecules maycomprise a fluorophore. In preferred embodiments, the donor and acceptormolecules comprise a fluorescent dye such 6-carboxyfluorescein (FAM,Amersham), 6-carboxy-X-rhodamine (REG, Amersham),N₁,N₁N¹,N¹-tetramethyl-6-carboxyrhodamine (TAMARA, Amersham),6-carboxy-X-rhodomine (ROX, Amersham), fluorescein, Cy5® (Amersham) andLightCycler-Red 640 (Roche Molecular Biochemicals). In a preferredembodiment, the donor molecules comprise FAM and the acceptor moleculescomprise REG, TAMARA or ROX. In an alternate embodiment, the donor isfluoroscein and the acceptor is Cy5® or LightCycler-Red 640 (RocheMolecular Biochemicals). Alternatively, the donor and acceptor moleculescomprise fluorescent labels such as the dansyl group, substitutedfluorescein derivatives, acridine derivatives, coumarin derivatives,pthalocyanines, tetramethylrhodamine, Texas Red®,9-(carboxyethyl)-3-hydroxy-6-oxo-6H-xanthenes, DABCYL®, BODIPY®(Molecular Probes, Eugene, Oreg.) can be utilized. Such labels areroutinely used with automated instrumentation for simultaneous highthroughput analysis of multiple samples.

[0023] Fluorescence monitoring of amplification is based on the conceptthat a fluorescence resonance energy transfer occurs between twoadjacent fluorophores and a measurable signal is produced. When anexternal light source, such as a laser or lamp-based system is applied,the donor molecule is excited and it emits light of a wavelength that inturn excites an acceptor molecule that is in close proximity to thedonor molecule. The acceptor molecule then emits an identifiable signal(i.e., a fluorescent emission at a distinct wavelength) that canmeasured and quantified. The donor molecule does not transmit a signalto acceptor molecules that are not in close proximity. Thus, when theddNTP incorporates into the primer, the donor and acceptor molecules arebrought close together and a fluorescence energy transfer occurs betweenthe two fluorophores causing the acceptor molecule to emit a detectablesignal. Acceptor molecules that are in close proximity to donor moleculeemit a signal that is distinctly different from the acceptor moleculesalone (i. e., an acceptor molecule that is not in proximity with thedonor). In addition, multiple different acceptor molecules may be used,in which each acceptor “combines” with the same donor molecule toproduce distinct signals, each being characteristic of a specificdonor-acceptor combination. Monitoring the fluorescence emission fromthe acceptor fluorophore after excitation of the donor fluorophoreallows highly sensitive product analysis.

[0024] Methods of the invention are especially useful to detect indiciaof cancer or precancer in a heterogeneous sample. Stool is a goodexample of a heterogeneous sample in which methods of the invention areuseful. A typical stool sample contains patient nucleic acids, but alsocontains heterologous nucleic acids, proteins, and other cellular debrisconsistent with the lytic function of the various nucleases, proteinasesand the like found in the colon. Under normal circumstances, stoolsolidifies as it proceeds from the proximal colon to the distal colon.As the solidifying stool passes through the colon, colonic epithelialcells are sloughed onto the stool. If a patient has a developing tumoror adenoma, cells from the tumor or adenoma will also be sloughed ontostool. Those cells, and/or their debris, will contain molecular indiciaof disease (e.g., mutations or loss of heterozygosity). In the earlystages of development, nucleic acid indicative of an adenoma or tumorcomprise only about 1% of the nucleic acid in a voided stool. If leftuntreated, proportionately more disease-related nucleic acids are foundin stool. Methods of the invention are useful for detecting early-stagelesions in heterogeneous samples such as stool. Methods of the inventionresult in a high degree of sensitivity and specificity for the detectionof early-stage disease. Methods of the invention are especially usefulin detecting, for example, adenomas in the colon. Adenomas arenon-metastatic lesions that frequently have the potential formetastasis. If all adenomas in a patient are detected and removed, theprobability of complete cure is virtually certain.

[0025] The methods of the present invention also exploit the discoverythat mutations in the BAT-26 segment of the MSH2 mismatch repair geneare closely associated with inherited cancers (and pre-cancerouslesions). In particular, BAT-26 mutations are highly-associated withHereditary Non-Polyposis Colorectal Cancer (“HNPCC”) (i.e., in greaterthan 90% of patients), making BAT-26 an ideal marker for screeningassays to detect this colorectal cancer, or colorectal adenoma that mayor may not develop into cancer. Use of methods of the invention on theBAT-26 locus identifies the characteristic deletions by producing anextension product in affected DNA that is shorter than the expectedwild-type extension product. Methods of the invention will beexemplified below using the BAT-26 locus. However, methods of theinvention are appreciated to be useful on any genetic locus in which adeletion occurs. Especially useful loci are those correlated withdisease, and especially cancer.

[0026] Furthermore, BAT-26 mutations have been found to be associatedwith cancers located in the right-hand (proximal) side of the colon.Thus, the methods of the present invention contemplate utilizing acombinatorial testing approach to screen patients, wherein BAT-26testing is used to screen the right side of the colon, and flexiblesigmoidoscopy is utilized to screen the left hand (distal/lower) side ofthe colon. Such a testing methodology permits a far more thorough screenfor cancerous and/or precancerous lesions than was previously possibleusing tests practiced in the art. Thus, in another embodiment, thepresent invention provides methods for detecting the presence ofcolorectal cancerous or precancerous lesions comprising (i) conductingin a sample obtained non-invasively or minimally-invasively from apatient an assay to identify a BAT-26 marker in the sample, and (ii)performing a flexible sigmoidoscopy on the patient.

[0027] The methods of the invention are useful for detecting diseases ordisorders related to the colon including, but not limited to, cancer,pre-cancer and other diseases or disorders such as adenoma, polyp,inflammatory bowel disorder, inflammatory bowel syndrome, regionalenteritis, granulomatous ileitis granulomatous ileocolitis, Crohn'sDisease, ileitis, ileocolitis, jejunoileitis, granulomatous colitis,Yersinia enterocolitica enteritis, ulcerative colitis,psuedo-membraneous colitis, irritable bowel syndrome, diverticulosis,diverticulitis, intestinal parasites, infectious gastroenteritis, toxicgastroenteritis, and bacterial gastroenteritis.

[0028] The methods of the present invention also provide for the use ofBAT-26 as a marker for detection of cancerous and precancerous lesionsby analysis of heterogeneous samples (e.g., stool). Such methodscomprise obtaining a representative sample of a stool voided by apatient and performing an assay on the sample to identify a BAT-26marker in the sample.

[0029] In another preferred embodiment, methods of the inventioncomprise selecting one or more mutational events that are indicative ofcancer, precancer, or other diseases or disorders, such that thecombined informativeness of the one or more events meets or exceeds apredetermined or desired level of informativeness. The informativenessof any mutation or combination of mutations may be validated by anaccepted invasive screening technique. For example, in methods to detectcolorectal cancer, the informativeness of a molecular assay may bedetermined by identification of a lesion using colonoscopy.

[0030] A detailed description of certain preferred embodiments of theinvention is provided below. Other embodiments of the invention areapparent upon review of the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031]FIG. 1A shows BAT-26 deletion detection using primer extensionreactions that incorporate labeled bases before the 3′ end of theextension product.

[0032]FIG. 1B shows BAT-26 deletion detection using primer extensionreactions that incorporate labeled bases at the 3′ end of the extensionproduct.

[0033]FIG. 2 shows deletion detection at the APC1309 locus.

[0034]FIG. 3 is a table showing the results of a clinical study ofscreening assays performed on 40 subjects using various markers,including BAT-26.

[0035]FIG. 4 is a pictorial representation of the location of nineteencancers located in the study described in FIG. 3.

[0036]FIGS. 5 and 6 are tables showing the results of a clinical studyof screening assays performed on 28 subjects using various markers,including BAT-26. FIG. 7 depicts the DNA sequence of the BAT-26 locus,wherein each “n” corresponds to a nucleotide of unknown identity.

DETAILED DESCRIPTION OF THE INVENTION

[0037] Methods of the invention provide non-invasive orminimally-invasive and highly sensitive assays for detecting thepresence of mutations in nucleic acid samples for the detection of earlystage cancer, precancer, or other diseases or disorders. Methods of theinvention also provide non-invasive or minimally invasive and highlysensitive assays for determining the presence of other indicia such asspecific alleles or variants in nucleic acid samples for the detectionof early stage cancer, precancer or other diseases or disorders. Methodsof the invention are especially useful for detecting the presence ofnucleic acid deletions and/or insertions in heterogeneous biologicalsamples to detect disease such as cancer or precancer. In preferredembodiments, methods of the invention are useful to detect mutations atloci that are associated with a disease such as cancer by identifying ina patient sample one or more nucleic acid mutations(s) that provide highsensitivity and high specificity for detection of the indicia of canceror precancer. Methods of the invention comprise identifying mutationshaving a known informativeness for cancer or precancer, or may be basedupon validating selected mutations or assays to detect them with respectto a standard assay for cancer. Preferred methods comprise assaysutilizing detection of BAT-26 mutations. By utilizing cancer orprecancer markers having a high sensitivity/specificity for detectingthe presence of cancer or precancer, methods of the invention provideimprovements in non-invasive or minimally-invasive molecular screeningassays. For purposes of the present invention, non-invasive orminimally-invasive indicates that specimens for analysis are selectedfrom the group consisting of stool, sputum, blood, urine, bile,cerebrospinal fluid, seminal fluid, saliva, aspirate, pancreatic juice,and the like. However, any tissue or body fluid specimen may be usedaccording to methods of the invention.

[0038] In general, methods of the invention comprise identifying atarget nucleic acid region that is suspected of being mutated, andinterrogating the target region using a primer extension reaction. Aprimer is hybridized upstream of the target region and extended throughthe target region. The extension reaction is terminated at a site beyondthe target region. The extension product is analyzed, and the size ofthe product is used as an indicator of the presence or absence of amutation in the target nucleic acid region. In general, the presence ofan extension product that is smaller than expected is indicative of thepresence of a deletion in the target region. Conversely, the presence ofa labeled extension product that is larger than expected is generallyindicative of the presence of an insertion in the target region.However, the presence of a small or large labeled extension product canalso be an indicator of a point mutation in the target region, asexplained in greater detail in the following sections.

[0039] Methods of the invention are particularly useful when the targetregion contains a sequence that causes the extending polymerase topause, stutter, or terminate prematurely. For example, regionscontaining nucleotide repeats such as a tract of a given nucleotide(such as the poly-A tract at the BAT-26 locus) dinucleotide ortrinucleotide repeats. However, the invention is generally useful todetect mutations at loci having a known wild-type nucleic acid.

[0040] In a preferred embodiment, a primer is hybridized upstream of atarget region that contains at most three different nucleotide bases.The hybridized primer is extended through the target region in thepresence of unlabeled nucleotides that are complementary to nucleotidesof the target region. The primer extension product is further extendedin the presence of a labeled terminator nucleotide that is complementaryto a nucleotide found downstream from the target region, but not foundin the target region. An extension product is only labeled if thelabeled terminator nucleotide is incorporated in the extension reaction.Consequently, an extension product is only labeled if it is extendedthrough the target region, and along to the template nucleotide that iscomplementary to the labeled terminator nucleotide. Accordingly,prematurely terminated extension products are not labeled and do notinterfere with the detection and analysis of labeled product.

[0041] The present invention comprises embodiments wherein the primer islabeled, or wherein a labeled nucleotide is incorporated into theextension product before extension through the target region iscomplete, provided that an additional label is incorporated into fullyextended products so that they can be distinguished from prematurelyterminated extension products. In one embodiment, a primer is labeledwith a first label, the labeled primer is hybridized upstream of thetarget region and extended through the target region, a second label isincorporated into the extension product downstream from the targetregion, and the extension reaction is terminated. Consequently, anextension product that terminates prematurely within the target regiononly contains the first label, whereas a fully extended product containsboth the first and second label. Accordingly, diagnostically relevantextension products are those that contain both labels.

[0042] Methods of the invention also comprise assays in which theextension product is labeled and terminated in separate steps, afterextension through the target region is complete. In one embodiment, atemplate nucleic acid comprises a target region consisting of a repeatof a first nucleotide base or a microsatellite region. Downstream fromthe target region is a second nucleotide base followed by a thirdnucleotide base. A primer is hybridized upstream of the target regionand extended through the target region in the presence of unlabelednucleotides that are complementary to the first nucleotide. Afterextension through the target region is complete, the extension productis further extended in the presence of a labeled nucleotide that iscomplementary to the second nucleotide of the template. Finally, thelabeled extension product is terminated via an extension reaction in thepresence of a terminator nucleotide (such as a dideoxy nucleotide) thatis complementary to the third nucleotide of the template. Otherembodiments of this aspect of the invention are also described in thefollowing sections.

[0043] Accordingly, an important aspect of the invention is a primerextension reaction wherein prematurely terminated extension products canbe distinguished from complete extension products that have notundergone premature termination. Preferably, prematurely terminatedextension products are not labeled, whereas complete extension productsare detectably labeled. FIGS. 1A and 1B the usefulness of the inventionin a deletion detection assay. The experimental details relating toFIGS. 1A and 1B are described in greater detail in Example 1. FIGS. 1Aand 1B show that the invention provides an effective method forminimizing background when interrogating a target nucleic acid regionsuspected of containing a deletion. FIG. 1A shows multiple samples thatwere analyzed by a primer extension assay that incorporated labelednucleotides into the extension product upstream of the target region. InFIG. 1B, the same samples were analyzed according to methods of theinvention. FIG. 1B does not contain the background of labeledprematurely terminated extension products that are seen in FIG. 1A.Consequently, the presence of a deletion is clearly indicated in lane 7of FIG. 1B, whereas lane 7 of FIG. 1A is more difficult to interpret.

[0044] Additional aspects of the invention are described in thefollowing sections and illustrated by the Examples.

[0045] Choosing the target region and the oligonucleotide primer

[0046] Preferably, a locus associated with a disease such as cancer ischosen. Most preferably, a locus that is known to frequently exhibit oneor more deletions is chosen. Useful loci include those containing atmost 3 out of the 4 possible nucleotide bases. Preferably, a chosenlocus comprises a polynucleotide region in which the deletion issuspected to occur. Once a locus is chosen, primers are designed orchosen to maximize specificity of binding to a nucleotide sequenceimmediately upstream of the region suspected of containing a deletion.The primer must hybridize immediately upstream of the region suspectedof containing the deletion so that no labeled nucleotide is incorporatedinto the primer extension product.

[0047] Sample preparation and hybridization

[0048] Methods of the invention are performed on any tissue or bodyfluid, including biopsy samples, and others having a high concentrationof affected (i.e., mutated) cells or cellular debris. However, methodsof the invention are particularly useful for detecting mutations inheterogeneous biological samples. A preferred sample is stool. For theanalysis of stool samples, preferred methods of the invention compriseobtaining at least a cross-section or circumferential portion of avoided stool as taught in U.S. Pat. No. 5,741,650, and co-pending,co-owned U.S. patent application Ser. No. 09/059,718, both of which areincorporated by reference herein. While a cross-sectional orcircumferential portion of stool is desirable, methods provided hereinare conducted on random samples obtained from voided stool, whichinclude smears or scrapings. Once obtained, the stool specimen ishomogenized. A preferable buffer for homogenization is one that containsat least 16 mM ethylenediaminetetraacetic acid (EDTA), as taught inco-pending, co-owned U.S. patent application Ser. No. 60/122,177,incorporated by reference herein. It has been discovered that the use ofat least 100 mM EDTA, and preferably 150 mM EDTA greatly improves theyield of nucleic acid from stool. Thus, a preferred buffer for stoolhomogenization comprises phosphate buffered saline, 20-100 mM NaCl orKCl, at least 100 mM EDTA, and optionally a detergent (such as SDS) anda proteinase (e.g., proteinase K).

[0049] After homogenization, nucleic acid is preferably isolated fromthe stool sample. Isolation or extraction of nucleic acid is notrequired in all methods of the invention, as certain detectiontechniques can be adequately performed in homogenized stool withoutisolation of nucleic acids. In a preferred embodiment, however,homogenized stool is spun to create a supernatant containing nucleicacids, proteins, lipids, and other cellular debris. The supernatant istreated with a detergent and proteinase to degrade protein, and thenucleic acid is phenol-chloroform extracted. The extracted nucleic acidsare then precipitated with alcohol. Other techniques can be used toisolate nucleic acid from the sample. Such techniques include hybridcapture, and amplification directly from the homogenized stool. Nucleicacids can be purified and/or isolated to the extent required by thescreening assay to be employed.

[0050] Nucleic acids to be analyzed are chosen based upon known orsuspected relationships between specific sequences and cancer orprecancer. Such sequences may comprise a mutation, or a specific alleleor variant. If desired, sequence-specific hybrid capture is used toisolate specific nucleic acids from the sample. Target nucleic acids maybe analyzed by any method of the art. Examples of preferred methodsinclude enumerative analysis of a loss of heterozygosity as taught inU.S. Pat. No. 5,670,325, incorporated by reference herein. Enumerativemethods compare the number in a sample of a wild-type nucleic acid knownnot to be altered in cancer or precancer with the number of a wild-typenucleic acid known or suspected to be altered in cancer or precancer. Astatistically-significant difference in the two numbers indicates apositive screen.

[0051] Target nucleic acids may also be analyzed by single baseextension techniques to identify, for example, a single nucleotidevariant or point mutation indicative of cancer or precancer. Preferably,single base extension assay are cycled as taught in co-owned, co-pendingU.S. patent application Ser. No. 09/067,212, incorporated by referenceherein. Briefly, cycled single base extension reactions compriseannealing a nucleic acid primer immediately 5′ to a region containing asingle base to be detected. The single base to be detected represents amarker for a mutation. The mutation may be, for example, a single pointmutation or a larger mutation for which the single base is a marker. Twoseparate reactions are conducted. In the first reaction, primer isannealed to target, and labeled (preferably with ³²P) nucleic acidscomplementary to non-wild type (e.g. mutants indicative of disease)variants at the single base to be detected, and unlabeled dideoxynucleic acids complementary to the wild-type base are combined. Primerextension is stopped the first time a wild-type (dideoxy) base is addedto the primer. Presence of label in the extended primer is indicative ofthe presence of a mutation. In a second reaction, the positive controlcontains labeled nucleic acid complementary to the wild-type base in thepresence of the primer. A DNA polymerase, such as Sequenase™ (Amersham),is used for primer extension. In a preferred embodiment, a thermostablepolymerase, such as Taq or thermal sequenase is used to allow moreefficient cycling. Once an extension reaction is completed, the firstand second probes bound to target nucleic acids are dissociated byheating the reaction mixture above the melting temperature of thehybrids. The reaction mixture is then cooled below the meltingtemperature of the hybrids and additional primer is permitted toassociate with target nucleic acids for another round of extensionreactions. In a preferred embodiment, 10 to 50 cycles of extensionreactions are conducted. In a most preferred embodiment, 30 cycles ofextension reactions are conducted. After completion of all cycles,extension products are isolated and detected. In alternativeembodiments, chain-terminating methods other than dideoxy nucleotidesmay be used. For example, chain termination occurs when no additionalbases are available for incorporation at the next available nucleotideon the primer.

[0052] Primer extension, labeling and termination

[0053] A hybridized primer is extended through the target region usingknown methods for primer extension, including extension using DNApolymerases. An extended primer preferably is labeled using a detectablelabel. Preferably, a labeled nucleotide is added to the extended primeronce extension through the target region is complete. In a preferredembodiment, the labeled extension reaction is terminated at apredetermined position downstream from the target region. In a preferredembodiment, the labeling and terminating steps are performedsimultaneously. In one embodiment, a labeled terminator nucleotide isincorporated into the extended primer downstream from the target region.Alternatively, the labeling and terminating steps are performedseparately. Preferably, the labeling and terminating reactions areperformed at about the same predetermined site downstream from thetarget region. If not, premature termination of a labeled extensionproduct can interfere with the analysis of the results. Indeed, if alabeled primer extension product must be extended significantly in orderto reach the predetermined termination site, then premature terminationof the labeled extension product results in a shorter than expectedlabeled extension product. This short extension product may result ineither a false positive indication of a deletion, or creates abackground that interferes with the detection of a short extensionproduct resulting from a deletion in the target region. Preferably thelabeled base is also a terminator base. More preferably the labeled baseis incorporated immediately upstream of the terminator base. The labelis preferably a radioactive isotope. Alternatively a fluorescent tag, amolecular weight tag or other detectable label may be used.

[0054] Detection and analysis of the extension product

[0055] While unlabeled primer extension products are contemplated, inpreferred methods of the invention, only extension products that havebeen extended through the region suspected of containing a deletion areanalyzed, because they are the only extension products that contain adetectable label. Extension products that terminate prematurely withinthe region suspected of containing a mutation are not labeled and arenot detected in the assay. Therefore, these premature extension productsdo not contribute to background noise that interferes with the analysisof the results.

[0056] Extended primer products are preferably detected using gelelectrophoresis, mass spectroscopy, sequencing, and other methods fordetermining the differential length of two primers.

[0057] The following examples illustrate practice of the invention usingdeletion detection in the BAT-26 and APC 1309 loci on samples preparedfrom stool specimens.

EXAMPLE 1 Deletion Detection at the BAT-26 Locus

[0058] Experiments were conducted to demonstrate the usefulness of theinvention to detect deletions in the BAT-26 locus. The followingexperiment compares the specificity for detecting deletions at theBAT-26 locus using primer extension reactions that incorporate labelbefore extension through the target region versus primer extensionreactions that incorporate label at the 3′ end of the extension product.

[0059] The nucleic acid template was prepared as follows. Templatenucleic acid containing the BAT-26 locus was amplified by PCR. To each50 μl PCR reaction tube, 40 μl of washed streptavidin coated Dynal beadswere added and mixed by vortexing on a high setting for a few seconds.The mixture was incubated in a rack at room temperature for 15 minutes,and mixed by vortexing after 5 minutes and 10 minutes of the incubationperiod. The tube was placed in a magnetic tube holder, and thesupernatant was removed. A 100 μl aliquot of 2×binding & wash buffer wasadded to each sample, and vortexed on a high setting for a few seconds.The tube was again placed in a magnetic tube holder and the supernatantwas removed. A 100 μl aliquot of 0.1 M NaOH was added to each tube, andmixed by vortexing on high for a few seconds. After a 5 minuteincubation at room temperature, the tubes were placed in a magnetic tubeholder, and the supernatant was removed. A further 100 μl of 0.1 M NaOHwas added, and vortexed for a few seconds. After placing the tube in amagnetic tube holder and removing the supernatant, 100 μl of 1×binding &washing buffer was added and vortexed for a few seconds on a highsetting. The tube was placed in a magnetic tube holder, the supernatantwas removed, and 100 μl of 1×TE pH 8.0 was added. The tube was vortexedon high for a few seconds, placed in a magnetic tube holder, and thesupernatant was removed. The beads were resuspended in 100 μl of 0.1×TEpH 8.0 buffer by vortexing on high for a few seconds. The resultingsamples were used in the assays, and may be stored at 4° C. for up to 1month.

[0060] In a first experiment, 5 μl of bead-bound PCR product was addedto the following primer extension reaction mixture: 9.625 μl of sterilemolecular biology grade diH₂0, 2.5 μl of 10×Sequenase Buffer, 2.5 μl of5 uM primer 1, 2.5 μl of 2 mM dATP, 2.5 μl of 50 μM ddGTP, 0.125 μl of³²P dTTP, and 0.25 μl of Sequenase.

[0061] The reaction mixture was cycled in an MJ Research TetradThermalcycler according to the following temperature profile.Temperature Time # Cycles 94° C. 5 min 1 94° C. 30 sec 52° C. 10 sec 3072° C. 10 sec  4° C. May be taken out of cycler immediately or afterovernight run

[0062] A 15 μl aliquot of formamide based stop solution was added toeach sample and mixed by pipetting up and down 5 times. A 7 μl aliquotfrom each sample was analyzed using a 15% denaturing polyacrylamide gelwith 7 M Urea in 1×TBE running buffer. The gel was dried and analyzedusing a Packard Instant Imager. Results are shown in FIG. 1A. Lanes 1-8are analyses of DNA obtained from patient stool samples. Lanes 9-14 arecontrols. Lane 9 contains no DNA template. Lanes 10, 13, and 14 contain,respectively, 0%, 1%, and 5% mutant DNA with a deletion within thepoly-A stretch of the BAT-26 locus. Lanes 11 and 12 are no PCR controls.

[0063] In a second experiment, 5 μl of bead bound PCR product was addedto the following primer extension reaction mixture: 7.125 μl of sterilemolecular biology grade diH20, 2.5 μl of 10×Sequenase Buffer, 2.5 μl of5 μM primer 2, 2.54 μl of 2 mM dATP, 2.5 μl of 50 μM ddTTP, 2.54 μl of0.1 μM dGTP, 0.125 μl of 32P dGTP, and 0.25 μl of Sequenase.

[0064] The reaction mixture was exposed to the same temperature cyclingas the reaction mixture in the first experiment, and the products wereseparated on a polyacrylamide gel under the same conditions. Lanes 1-14of FIG. 1B show results of this second experiment. The same nucleic acidtemplates were used in the reactions shown in lanes 1-14 of FIG. 1A andlanes 1-14 of FIG. 1B.

[0065] In the first experiment, shown in FIG. 1A, the radioactive dGTPwas incorporated into the primer extension product before it wasextended through the poly-A stretch of the BAT-26 locus. Primer 1(5′-AGCCCTTAACCTTTTTCAGG-3′, SEQ ID No: 1) used in the first experiment,hybridizes immediately upstream of a site where dTTP is incorporated (anA on the template strand). Accordingly, prematurely terminated extensionproducts are labeled and appear as background in all of lanes 1-8.

[0066] In the second experiment, shown in FIG. 1B, the radioactive dTTPwas incorporated into the primer extension product after it was extendedthrough the poly-A stretch of the BAT-26 locus. The 3′ end of primer 2(5′-GCCCTTAACCTTTTTCAGGT-3′, SEQ ID NO: 2) used in the secondexperiment, includes the T that is immediately downstream from primer 1.Accordingly, in the second reaction, radioactive dTTP is onlyincorporated into the primer extension product after it has beenextended through the poly-A stretch. Furthermore, the extension reactionis also terminated close to the site of ³²P dGTP incorporation. Thesecond reaction mixture also contains ddTTP, and some of the extensionproducts incorporate ³²PdGTP followed by ddTTP at the T repeatdownstream from the poly-A or microsatellite stretch. Accordingly, inthe second experiment, primer extension products that terminateprematurely within the poly-A stretch are not labeled and are not seenas background in lanes 1-8, nor in control lanes 9-14. In FIG. 1B, onlylanes 6 and 7, and control lanes 13 and 14, contain short labeled primerextension product. The only samples that contained nucleic acid templatehaving a deletion in the poly-A stretch were the ones that were analyzedin lanes 6, 7, 13, and 14. The sample of lane 6 was contaminated with asmall amount of deleted template. The sample of lane 7 was from apatient with colon cancer associated with a deletion in the poly-Astretch of the BAT-26 locus. The samples of lanes 13 and 14 contained 1%and 5% mutant DNA, respectively.

[0067] A comparison of FIGS. 1A and 1B, shows that methods of theinvention reduce the background of primer extension reactions. As aresult, the analysis is much easier to interpret. Indeed, the presenceof smaller than expected extension products in the second experiment isan indication of the presence of mutant nucleic acid in the sample. Inthe first experiment, smaller than expected extension products arepresent in all reactions, and the analysis is more complicated.

[0068] In addition, methods of the invention, illustrated by the resultsof the second experiment, can be used to detect a very small amount ofmutant nucleic acid in a heterogeneous sample containing mainly normalnucleic acid. The results shown in lanes 6 and 13 are the most striking.In FIG. 1A, it is difficult to decide whether a deletion product ispresent in lanes 6 and 13. In contrast, a deletion product is clearlypresent in lanes 6 and 13 of FIG. 1B.

[0069] Methods of the invention are particularly useful for analyzingloci such as BAT-26, where a stretch of repeated nucleotide sequenceinterferes the with efficient extension of DNA polymerase reactions.Premature termination of extension reactions is typically more frequentat such loci.

EXAMPLE 2 Deletion Detection at the APC 1309 Locus

[0070] A deletion of 5 nucleotides is often found at codon 1309 of theAPC gene. The nucleotide sequence at this location is 5′-GAAAAGATT-3′(SEQ ID NO: 3) in the wild-type gene. Typical deletions consist of GAAAA(SEQ ID NO: 4), AAAAG (SEQ ID NO: 5), or AAAGA (SEQ ID NO: 6). To detectany of these deletions using a method of the invention, a 17-baseoligonucleotide was designed to hybridize immediately upstream of theposition of the first G (the G of the GAA codon above). Hybridizedprimer was extended in the presence of unlabeled dATP, unlabeled dGTP,and ³²P-ddTTP. Accordingly, the extension product is only labeled if itis extended through the target region suspected of containing a deletionand the labeled ddTTP is incorporated. The expected wild-type product is25 bases long, whereas any of the deletions described above generates a20-base long extension product.

[0071] The extension reaction was performed on a duplicates of patientsamples and the results are shown in FIG. 2. Controls containing 0%, 1%,and 5% mutant nucleic acid were also analyzed that contained a 5 basepair deletion in BAT-26. The control results indicate that the presenceof 1% mutant nucleic can be detected unambiguously. Both tests forpatient #508 indicated the presence of a deletion at the 1309 locus.Patient #508 did indeed have colon cancer associated with a deletion atthe 1309 locus.

[0072] In contrast, the results for patients without a deletion at the1309 locus showed no background at the position characteristic of adeletion containing extension product. Accordingly, methods of theinvention are useful for a simple test for the presence of a deletion atthe 1309 locus.

[0073] The invention will be exemplified further with experiments todetect the presence of indicia of colorectal cancer or precancer insamples prepared from patient stool specimens. However, the skilledartisan recognizes that methods of the invention can be practiced usinga variety of different samples in order to detect a variety of cancers,pre-cancers, and other diseases and disorders.

[0074] A reason that detection of colorectal cancer or precancer (e.g.,an adenoma) is exemplified is that a stool specimen is a good example ofa heterogeneous environment in which methods of the invention areespecially useful (see above). Moreover, colonoscopy (and sigmoidoscopy,a related technique) is a well-known invasive standard that has a highsensitivity and high specificity (although high cost and low patientcompliance) with which methods of the invention can be compared andvalidated.

[0075] Methods of the invention comprise screening a sample, such as oneprepared from a stool specimen, for the presence of one or moremarker(s) of cancer, precancer, or other diseases or disorders (e.g., acolorectal tumor or adenoma), such that the sensitivity of detection isbetween about 50% and about 100%, and the specificity of detection isbetween about 85% and about 100%. In a preferred embodiment, methods ofthe invention combine different types of assays in order to achieve anoverall increase in sensitivity and specificity. Thus, methods of theinvention comprise conducting an assay for a mutation known to beassociated with cancer, precancer or another disease or disorder, and anassay for a quantity and/or length of DNA expected to occur inconjunction with the cancer, precancer, or other disease or disorder inorder to obtain the combined benefits of the sensitivity and specificityof both assays. Moreover, embedded within the concept of utilizingmultiple nucleic acid analyses to detect a disease or disorder is theuse of multiple genomic targets in each assay in order to providefurther increases in sensitivity and specificity. However, as shownbelow, a single-marker assay is sufficient for practice of theinvention.

[0076] The genomic targets and assay methods used according to theinvention can vary depending upon the desired level of sensitivity andspecificity, as well as the type of disease or disorder the detection ofwhich is desired. Genomic targets (e.g., mutations) are selected basedupon their known sensitivity or specificity or by determining a baselinesensitivity and specificity. In preferred embodiments, methods of theinvention comprise the detection of a mutation at a single, informativelocus. In other embodiments, assays for informative loci are combined inorder to achieve improved sensitivity and specificity of detectionrelative to invasive techniques. Accordingly, methods of the inventioncontemplate a combination of assays selected from multiple mutationdetection, quantitative polymerase chain reaction (i.e., to determinethe amount of amplifiable DNA in a sample), sequence-specific hybridcapture, oligo-ligation, amplification refractory mutation system,single-stranded conformational polymorphism detection, sequencing,mismatch detection, and single base extension. Target loci includechromosomes 1, 5, 8, 17, and 18, particularly chromosome 5q, chromosome17p, chromosome 8p, chromosome 1q, and chromosome 18q. Preferred locifor use in methods of the invention include p53, APC, BAT-26, and otherssuspected to be predictive of certain diseases or disorders. A preferredlocus for use in methods of the invention is BAT-26.

[0077] Other genes are known to be associated with colorectal cancer,and their sensitivity and specificity are determined when not known inthe literature by determining the percentage of tumors bearing themutation, and the percentage of healthy specimens that bear the mutationfrom a sufficiently large and diverse population. This can be doneempirically, or mathematically using algorithms that predict thelikelihood of false positive and false negative screening results basedupon data relating the presence of a mutation to the presence of cancer,pre-cancer or another disease or disorder. In the case of colorectalcancer, confirmation of a patient's clinical status can be accomplishedby a standard test such as colonoscopy (which has a typical sensitivityof 95% and a typical specificity of 100%). The preferred method ofanalysis of stool samples, as discussed earlier, comprises obtaining atleast a cross-section or circumferential portion of a voided stool.While a cross-sectional or circumferential portion of stool isdesirable, methods provided herein are conducted on random samplesobtained from voided stool, which include smears or scrapings. Onceobtained, the stool specimen is homogenized in a buffer that contains atleast 16 mM ethylenediaminetetraacetic acid (EDTA). However, asdiscussed earlier, it has been discovered that the use of at least 150mM EDTA greatly improves the yield of nucleic acid from stool.

[0078] Methods of the invention are also useful for screeningpopulations of patients in order to identify characteristics inpopulation samples that are indicative of cancer or adenoma. Forexample, methods of the invention comprise high sensitivity, highspecificity screening of populations of patients in order to correlatenucleic acid mutations or polymorphic variants present in a subset ofpatient samples with the presence of disease in those patients. Thus,methods of the invention comprise detecting genomic variations inpatient samples, correlating those variations with confirmed disease,and using the variations associated with confirmed disease as adiagnostic screen for the disease in subsequent patient samples. Suchmethods preferably are performed on pooled samples, such as stoolsamples, from identified populations of patients (e.g., diseased,healthy). Such methods are preferably based upon variations in singlenucleotide polymorphic loci. The sensitivity and specificity ofdetecting variants in those loci as a function of disease is determined.Those loci that predict disease at predefined levels of sensitivity andspecificity are selected for use in screening assays for unknown patientsamples.

[0079] BAT-26 mutations have also been found to be associated withcancers located in the right-hand (proximal) side of the colon. Thus,the methods of the present invention contemplate the use of acombinatorial testing approach to screen patients, wherein BAT-26testing is used to screen the right side of the colon, and flexiblesigmoidoscopy is utilized to screen the left hand (distal/lower) side ofthe colon. This type of testing methodology permits a far morecomprehensive screen for cancerous and/or precancerous lesions than waspracticed previously in the art.

[0080] Methods of the invention are useful not only for detecting canceror precancer, but also for detecting other colonic diseases or disordersthat may be correlated with specific nucleic acid markers including, butnot limited to, adenoma, polyp, inflammatory bowel disorder,inflammatory bowel syndrome, regional enteritis, granulomatous ileitisgranulomatous ileocolitis, Crohn's Disease, ileitis, ileocolitis,jejunoileitis, granulomatous colitis, Yersinia enterocolitica enteritis,ulcerative colitis, psuedo-membraneous colitis, irritable bowelsyndrome, diverticulosis, diverticulitis, intestinal parasites,infectious gastroenteritis, toxic gastroenteritis, and bacterialgastroenteritis.

[0081] The following examples provide further specific exemplificationof the concepts discussed above. The assays exemplified below are forpurposes of illustration.

EXAMPLE 3 Clinical Study of Cancer Detection Using BAT-26 Marker

[0082] Stool specimens were collected from 40 individuals who presentedat the Mayo Clinic (Rochester, Minn.) with symptoms or historyindicating that a colonoscopy should be performed. Each stool sample wasfrozen. Immediately after providing a stool sample, all individuals weregiven a colonoscopy in order to determine their disease status.Colonoscopy, an invasive test requiring sedation of the patient, has asensitivity approaching 95%, and a specificity of nearly 100% for thediagnosis of colonic neoplasia. Based upon the colonoscopy results andsubsequent histological analysis of biopsy samples taken duringcolonoscopy, individuals were placed into one of three groups: normal,cancer, and adenoma. An adenoma, or polyp, is considered clinicallyrelevant if it has a diameter of 1 cm or greater. Thus, all individualsin the adenoma group had a polyp of at least 1 cm in diameter. Patientsin the cancer group had tumors diagnosed as cancer, and the disease-freeindividuals were those for whom colonoscopy showed no sign of cancer oradenoma. Based upon the colonoscopy results, 21 patients were diagnosedwith cancer, 9 patients were diagnosed with an adenoma greater than 1cm, and 10 patients were free of cancer or adenoma.

[0083] Multiple mutation analysis was then performed, on a blinded basis(i.e., scientists performing the assays did not know the results ofcolonoscopy or histology), on each sample. Each frozen stool specimen,weighing from 7-33 grams, was thawed, homogenized in 500 mM Tris, 16 mMEDTA, and 10 mM NaCl, pH 9.0, at a volume to mass ratio of about 3:1.Samples were then rehomogenized in the same buffer to a finalvolume-to-mass ratio of 20:1, and spun in glass macro beads at 2356×g.The supernatant was collected and treated with SDS and proteinase k. TheDNA was then phenol-chloroform extracted and precipitated with alcohol.The precipitate was suspended in 10 mM Tris and 1 mM EDTA (1×TE), pH7.4. Finally, the DNA was treated with Rnase.

[0084] Human DNA was isolated from the precipitate by sequence-specifichybrid capture. Biotynilated probes against portions of the p53, K-ras,and APC genes were used.

[0085] A 10 μl aliquot of each probe (20 pmol/capture) was added to asuspension containing 300 μl DNA in the presence of 310 μl 6M GITCbuffer for 2 hours at room temperature. Hybrid complexes were isolatedusing streptavidin-coated beads (Dynal). After washing, probe-beadcomplexes were suspended at 25° C. for 1 hour in 0.1×TE buffer, pH 7.4.The suspension was then heated for 4 minutes at 85° C., and the beadswere removed.

[0086] Captured DNA was then amplified using PCR, essentially asdescribed in U.S. Pat. No. 4,683,202, incorporated by reference herein.

[0087] Samples were heated to 94° C. for 5 minutes, and then 40 cycleswere conducted between 94° C., 60° C., and 72° C. (1 minute each),followed by one cycle at 72° C. for 5 minutes.

[0088] Amplified nucleic acid samples were then run on an electophoreticgel and size differences in the amplified PCR products were observed todetect mutant samples.

[0089] As shown in FIG. 3, four out of nineteen cancers found had BAT-26mutations.

[0090] As shown in FIG. 4, all nineteen cancers were found in varyingparts of the colon, but only the right-sided cancers had BAT-26mutations.

EXAMPLE 4 Diagnostic Assay Using BAT-26

[0091] The BAT-26 mismatch repair locus (FIG. 7) was used to assess thesame 40 samples described above. Deletions in BAT-26 have beenassociated with colorectal cancer or adenomas. Samples were prepared asdescribed above. A primer was hybridized to the portion of the BAT-26locus immediately upstream of the poly-A tract, which consists of 26adenosines (nucleotides 195-221). Unlabeled deoxythymidine, a mixture oflabeled and unlabeled deoxycytosine, and unlabeled dideoxyadenine wereadded along with polymerase. The primer was extended through the poly-Aregion. The labeled and unlabelled cytosine was extended for the nextthree bases (nucleotides 222-224, all guanines in the intact sequence)such that label was incorporated into each extended primer. After thepoly-A tract and the three guanines, there exist two thymidines in theintact sequence. Thus, the dideoxyadenosine stops primer extension byaddition at the end of a primer that has been extended through thepoly-A and triguanine regions. Strands were separated, and the length ofthe strands was observed on a polyacrylamide gel to detect deletions inthe poly-A tract. The results are presented below in Table A: TABLE ADiagnosis Sensitivity Specificity Diagnosis By of of By BAT-26 BAT-26BAT-26 Patient Status Colonoscopy Detection Detection DetectionCancer/Adenoma 21/9 4/0 19%/0% 100%/0%

[0092] As shown above, BAT-26 alone did not provide the high sensitivityachieved using multiple mutation or quantitation alone, but showed highsensitivity in comparison with other single locus detection assays.Moreover, as shown below, BAT-26 in combination with the othertechniques described above produced an overall increase in sensitivityand specificity.

EXAMPLE 5 Cumulative Effects of Kras, Multiple Mutation, Quantitation,and BAT-26

[0093] The results obtained above for Kras, multiple mutation analysis,quantitation, and BAT-26 were combined to determine the cumulativeeffects of using combinations of those techniques in order to produceincreased sensitivity and specificity in a non-invasive assay for canceror precancer. The results are summarized below in Table B: TABLE B Krasand Quantitation Multiple Mutation Quantitation and and and QuantitationAssay Combination BAT-26 BAT-26 and BAT-26 Sensitivity for 80%/56%80%/56% 90%/78% Detection of Cancer/Adenoma Specificity for 100% 100%100% Detection of Cancer/Adenoma

[0094] As shown in the summary above, the combination of multiplemutation analysis, quantitative PCR, and BAT-26 produced a sensitivityapproaching that of colonoscopy. A combination of multiple mutationanalysis and quantitation alone also produces very high sensitivities.All assays resulted in a specificity of 100% (no false positiveresults), which is comparable to colonoscopy.

[0095] The foregoing experiments show that even a singlehigh-sensitivity/high specificity non-invasive or minimally-invasiveassay produces diagnostic results that are superior tonon-invasive/minimally-invasive techniques of the art, and approachresults observed with the recognized standard invasive diagnosticprocedure (colonoscopy). Moreover, a non-invasive assay utilizing morethan one high-sensitivity/high-specificity technique results indiagnostic accuracy approaching 100%. As such, methods of the inventionprovide a significant improvement in the ability to perform accuratenon-invasive diagnosis of cancer.

EXAMPLE 6 Clinical Study of Cancer Detection Using BAT-26 Marker

[0096] The methods described above in Example 3 were followed usingstool specimens collected from 28 individuals at the Mayo Clinic(Rochester, Minn.) with symptoms or history indicating that acolonoscopy should be performed. The results are shown in FIGS. 5 and 6,and demonstrated that the study found two of eight cancers with BAT-26mutations.

What is claimed is:
 1. A method for detecting a nucleic acid insertionor deletion, the method comprising the steps of: (a) selecting a nucleicacid having a known wild-type sequence and having a target regioncomprising a repeat sequence having at most three different types ofnucleotide bases selected from the group consisting of dGTP, dATP, dTTP,and dCTP; (b) contacting a sample with an oligonucleotide primer that iscomplementary to a portion of said nucleic acid immediately upstream ofsaid target region; (c) extending said primer in the presence ofnucleotide bases that are complementary to the nucleotide bases of thetarget region, thereby to form a primer extension product; (d) extendingthe primer extension product in the presence of a labeled nucleotidecomplementary to a nucleotide base downstream from the target region insaid nucleic acid, wherein said labeled nucleotide is not complementaryto any of the nucleotide bases of the target region, thereby to producea labeled extension product comprising a sequence that is complementaryto the entire target region; (e) detecting the labeled extensionproduct; and (f) comparing the size of the labeled extension productdetected in step e) to a standard, wherein a labeled extension productsmaller than the standard is indicative of the presence of a deletion inthe target region and a labeled extension product larger than thestandard is indicative of the presence of an insertion in the targetregion.
 2. The method of claim 1 , further comprising the step ofterminating the primer extension product by incorporating a terminatornucleotide in said product that is complementary to a nucleotidedownstream from the target region in a wild type nucleic acid, whereinsaid terminator nucleotide is not complementary to any of thenucleotides of the target region, said step of terminating the primerextension product being performed simultaneously with or immediatelyafter step (d).
 3. The method of claim 2 , wherein the labelednucleotide and the terminator nucleotide are the same.
 4. The method ofclaim 1 , wherein the labeling reaction of step (d) is performed in thepresence of labeled nucleotide and unlabeled nucleotide of the sametype.
 5. The method of claim 4 , wherein the ratio of labeled nucleotidebase to unlabeled nucleotide base is 1:1.6 (unlabeled:labeled).
 6. Themethod of claim 4 , wherein more than one nucleotide from step (d) isincorporated into the labeled extension product.
 7. The method of claim1 , wherein said sample contains a heterogeneous mixture of mutantnucleic acid having a deletion in the target region and wild typenucleic acid with no deletion in the target region.
 8. The method ofclaim 1 , wherein said sample is selected from the group consisting ofstool, homogenized stool, urine, semen, blood, saliva, sputum,cerebrospinal fluid, pancreatic juice, pus, and aspirate.
 9. The methodof claim 1 , wherein a deletion in the target region is indicative ofthe presence of cancerous or precancerous tissue in the biologicalsample.
 10. The method of claim 1 , wherein said sample includes abuffer comprising at least 100 mM EDTA.
 11. The method of claim 1 ,wherein said target region is the poly-A tract at the BAT-26 locus. 12.The method of claim 1 , wherein said target region is a microsatelliteregion.
 13. The method of claim 1 , wherein the presence of a deletionin said target region is associated with the presence of a mutation at aseparate genetic locus selected from the group consisting of APC, DCC,P53, and RAS.
 14. A method for detecting a nucleic acid deletion in asample, the method comprising the steps of: a) selecting a nucleic acidwith a known wild-type sequence and having a target region suspected ofcontaining a deletion, wherein said target region comprises a repeatsequence and contains at most three different types of nucleotidesselected from the group consisting of dGTP, dATP, dTTP, and dCTP; b)hybridizing an oligonucleotide primer to a region upstream of saidtarget region, in a nucleic acid sample; c) contacting said hybridizedoligonucleotide primer with an extension reaction mixture comprising: i)the nucleotides that are complementary to the nucleotides in the targetregion, ii) a labeled nucleotide that is complementary to a nucleotidefound downstream from the target region, but is not complementary to anynucleotide found within the target region, and iii) a terminatornucleotide that is complementary to a nucleotide found downstream fromthe target region, but is not complementary to any nucleotide found inthe target region; d) extending the hybridized oligonucleotide primer togenerate a labeled extension product comprising a sequence that iscomplementary to the entire target region; e) detecting the labeledextension product; and f) comparing the size of the labeled extensionproduct detected in step e) to a standard, wherein a labeled extensionproduct smaller than the standard is indicative of the presence of adeletion in the target region, and a labeled extension product largerthan the standard is indicative of the presence of an insertion.
 15. Themethod of claim 14 , wherein the target region is greater than fivebases long.
 16. The method of claim 14 , wherein the deletion orinsertion is greater than three bases long.
 17. The method of claim 14 ,wherein the standard is the wild-type labeled extension product and is amolecular weight standard.
 18. The method of claim 14 , wherein saidsample includes a buffer comprising at least 100 mM EDTA.
 19. A methodfor diagnosing colorectal cancer or precancer, the method comprising thesteps of: performing an assay to detect, in a stool sample from apatient, a nucleic acid mutation indicative of a colorectal lesion;performing a sigmoidoscopy on said patient; and diagnosing colorectalcancer or precancer in said patient if at least one of said assay andsaid sigmoidoscopy is positive.
 20. The method of claim 19 , whereinsaid assay is conducted prior to said sigmoidoscopy.
 21. The method ofclaim 19 , wherein said sigmoidoscopy is performed prior to said assay.22. The method of claim 19 , wherein said mutation is indicative of thepresence of a colorectal lesion in the proximal colon.
 23. The method ofclaim 19 , wherein said sample includes a buffer comprising at least 100mM EDTA.
 24. A method for localizing a colorectal lesion in a patient,the method comprising the steps of: performing an assay to detect, in astool sample from a patient, a nucleic acid mutation indicative of saidcolorectal lesion; performing a sigmoidoscopy on said patient;diagnosing a proximal colonic lesion if said assay is positive for themutation and said sigmoidoscopy is negative; and diagnosing a distalcolonic lesion if said sigmoidoscopy is positive and said assay isnegative for the mutation.
 25. A method for diagnosing hereditarynon-polyposis colorectal cancer, the method comprising the steps of:performing an assay to detect, in a stool sample from a patient, anucleic acid mutation indicative of said hereditary non-polyposiscolorectal cancer; performing a colonoscopy on said patient; anddiagnosing hereditary non-polyposis colorectal cancer if said assay ispositive and said colonoscopy reveals an adenoma.
 26. A method fordetermining whether a target nucleotide is present at a genetic locus ofinterest, the method comprising the steps of: (a) contacting a nucleicacid in a biological sample with a primer that is complementary to aportion of a genetic locus immediately upstream of a target nucleotideposition; (b) extending said primer in the presence of a labelednucleotide that is complementary to the target nucleotide; (c) furtherextending said primer in the presence of a terminator nucleotide that iscomplementary to a nucleotide downstream from the target nucleotide, butis not complementary to the target nucleotide, thereby to generate anextension product; and (d) determining whether said labeled nucleotideis present in said extension product, thereby to determine whether saidtarget nucleotide is present at said genetic locus.
 27. The method ofclaim 26 , wherein said biological sample is selected from the groupconsisting of stool, homogenized stool, urine, semen, blood, saliva,sputum, cerebrospinal fluid, pancreatic juice, pus, and aspirate. 28.The method of claim 26 , wherein said genetic locus comprises a geneselected from the group consisting of BAT-26, APC, DCC, P53, and RAS.29. The method of claim 26 , wherein said labeled nucleotide comprisesan acceptor molecule, and said primer comprises a donor molecule, saiddonor molecule being capable of activating said acceptor molecule so asto produce a detectable signal.
 30. The method of claim 26 , whereinsaid signal is a fluorescent signal characteristic of an activation ofthe acceptor molecule by the donor molecule.
 31. The method of claim 26, wherein said extensions are catalyzed by a thermostable polymerase.32. The method of claim 26 , wherein said genetic locus comprises amicrosatellite region, and said target nucleotide is in saidmicrosatellite region.
 33. A method for determining whether a targetpoint mutation is present at a genetic locus of interest, the methodcomprising the steps of: (a) contacting a nucleic acid in a biologicalsample with a primer that is complementary to a portion of a geneticlocus immediately upstream of a target point mutation position; (b)extending said primer in the presence of a labeled nucleotide that iscomplementary to a target nucleotide suspected to be present at saidtarget position; (c) further extending said primer in the presence of aterminator nucleotide that is complementary to a nucleotide downstreamfrom the target nucleotide, but is not complementary to the targetnucleotide, thereby to generate an extension product; and (d)determining whether said labeled nucleotide is present in said extensionproduct, thereby to determine whether said target point mutation ispresent at said genetic locus.
 34. The method of claim 33 , wherein saidpoint mutation is a transversion mutation.
 35. The method of claim 33 ,wherein said point mutation is a transition mutation.
 36. A method foridentifying a target single nucleotide polymorphic variant present at agenetic locus of interest, the method comprising the steps of: (a)contacting a nucleic acid in a biological sample with a primer that iscomplementary to a portion of said genetic locus immediately upstream ofa target single nucleotide polymorphic variant position; (b) extendingsaid primer in the presence of at least a first and a seconddifferentially labeled nucleotide, said first labeled nucleotide beingcomplementary to a first nucleotide suspected to be present at saidtarget position, said second labeled nucleotide being complementary to asecond nucleotide alternatively suspected to be present at said targetposition; (c) further extending the primer in the presence of aterminator nucleotide that is complementary to a nucleotide downstreamfrom the target position, wherein said terminator nucleotide is notcomplementary to said first or second nucleotides, thereby to produce anextension product; and (d) determining the identity of the labelednucleotide present in said extension product, thereby to determine theidentity of the target single nucleotide polymorphic variant present atthe genetic locus.
 37. The method of claim 36 , wherein said firstlabeled nucleotide comprises a first acceptor molecule, said secondlabeled nucleotide comprises a second acceptor molecule, said firstacceptor molecule being different from said second acceptor molecule,and said primer comprises a donor molecule being capable of activatingsaid first and second acceptor molecules so as to produce a first and asecond detectable signal, respectively.
 38. The method of claim 37 ,wherein said first and second detectable signals are differentfluorescent signals.
 39. A method for determining whether a targetsingle nucleotide polymorphic variant is present at a genetic locus ofinterest, the method comprising the steps of: (a) contacting a nucleicacid in a biological sample with a primer that is complementary to aportion of a genetic locus immediately upstream of a target singlenucleotide polymorphic variant position; (b) extending said primer inthe presence of a labeled nucleotide that is complementary to anucleotide suspected to be present at said target position; (c) furtherextending the primer in the presence of a terminator nucleotide that iscomplementary to a nucleotide downstream from said target nucleotide,wherein said terminator nucleotide is not complementary to said targetnucleotide, thereby to produce an extension product; and (d) determiningwhether said labeled nucleotide is present in said extension product,thereby to determine whether said target single nucleotide polymorphicvariant is present at said genetic locus.
 40. A method for determiningwhether a target nucleotide deletion is present at a genetic locus ofinterest, the method comprising the steps of: (a) contacting a nucleicacid in a biological sample with a primer that is complementary to aportion of a genetic locus immediately upstream of a target nucleotidedeletion position; (b) extending said primer in the presence of alabeled nucleotide that is complementary to the target nucleotide; (c)further extending said primer in the presence of a terminator nucleotidethat is complementary to a nucleotide downstream from the targetnucleotide, but is not complementary to the target nucleotide, therebyto generate an extension product; and (d) determining whether saidlabeled nucleotide is present in said extension product, thereby todetermine whether said target nucleotide deletion is present at saidgenetic locus.
 41. A method for determining whether a target nucleotideinsertion is present at a genetic locus of interest, the methodcomprising the steps of: (a) contacting a nucleic acid in a biologicalsample with a primer that is complementary to a portion of a geneticlocus immediately upstream of a target nucleotide insertion position;(b) extending said primer in the presence of a labeled nucleotide thatis complementary to the target nucleotide; (c) further extending saidprimer in the presence of a terminator nucleotide that is complementaryto a nucleotide downstream from the target nucleotide, but is notcomplementary to the target nucleotide, thereby to generate an extensionproduct; and (d) determining whether said labeled nucleotide is presentin said extension product, thereby to determine whether said targetnucleotide insertion is present at said genetic locus.
 42. A method forquantifying the number of a nucleic acid having a target nucleotidepresent at a genetic locus of interest, the method comprising the stepsof: (a) contacting a nucleic acid in a biological sample with a primerthat is complementary to a portion of a genetic locus immediatelyupstream of a target nucleotide position; (b) extending said primer inthe presence of a labeled nucleotide that is complementary to targetnucleotide, (c) further extending the primer in the presence of aterminator nucleotide that is complementary to a nucleotide downstreamfrom the target nucleotide, wherein said terminator nucleotide is notcomplementary to said target nucleotide, thereby to form an extensionproduct; and (d) enumerating the number of extension products thatcomprise the labeled nucleotide, thereby determining the number ofnucleic acids having the target nucleotide at the genetic locus.
 43. Themethod of claim 42 , wherein the amount of nucleotide present is anindicia of the severity of disease in a patient.